In this article our technical expert, Dr. Dawn Watson, will cover the polarity of the stationary phase, the column length, internal diameter, thickness of the stationary phase film and the required upper operating temperature.
The required time and conditions actually depend upon the polarity of the stationary phase (more polar phases bleed more, Figure 1), the column length, internal diameter and thickness of the stationary phase film (longer columns with thicker films and larger internal diameters mean more stationary phase to bleed) and the required upper operating temperature (i.e. the top temperature in the analytical method to be used).
Figure 1: Typical capillary GC stationary phase bleed profile for two different polysiloxane based stationary phases (Figure reproduced with permission, Agilent Technologies, Santa Clara, CA, USA).
The following steps should be followed to install and condition the GC column:
1. Connect the column to the GC inlet using good practice (clean cut, no finger marks, no shards of septum or ferrule stuck into the column inlet, column properly positioned in the inlet) â the inlet should be set at the temperature specified in the analytical method, the oven at ambient temperature, and the carrier gas should be flowing at the conditions stipulated in Table 1 below.
2. Connect the column to the detector unless you do not want bleed products to enter the detector (if MS detection used, or in line with your local policies and procedures).
3. Under these conditions allow at least six column volumes of carrier gas to pass through the column in order to remove air from the column as well as sparging any dissolved oxygen from within the stationary phase. This will allow you to condition the column for less time and at a lower temperature. To calculate the time for six column volumes to flow through the column use the following equations, or use the guidelines given in Table 1.
Where:
dc = column diameter (mm)
L = column length (mm)
For a 30 m x 0.25 mm column:
Therefore 6 column volumes = 1.473 x 6 = 8.84 mL
or 8.84 mins. at a carrier flow of 1.0 mL/min.
4. Once this step 3 completed, ramp the oven temperature (ensure the carrier gas is still flowing through the column) at 20 °C/min. to 20 °C above the upper temperature required by the analytical method. Once the upper temperature limit has been reached the column should be conditioned for the correct amount of time based on the dimensions and phase type (Table 2).
5. Once conditioned the column should be returned to the initial temperature required for the analysis and a visual assessment of baseline noise and drift made in order to confirm that the column is ready for use. System suitability standards may also be employed at this stage to make a quantitative assessment of fitness for purpose via Limit of Detection and Signal to Noise measurements.
Table 1: GC column purge times.
Table 2: Recommended conditioning times for various capillary GC column types.
As a brief aside, baseline rise can also be caused by running in constant carrier gas pressure mode with mass-flow sensitive detectors (such as FID) and one should also consider this fact as a contributor to baseline displacement alongside column bleed.
Finally, it is always good policy to record the bleed profile for a column when new (Figure 1), so that the level of bleed can be referenced at a later date in order to assess the degradation of the stationary phase over time and perhaps a performance limit established for column replacement. Simply run the method without making an injection or inject a small amount of sample solvent.
If using mass spectral detectors, the presence of ions at m/z 207, 281, and 355 indicate column bleed. These will almost always be there â it is whether they are causing problems either spectrally or in terms of reproducibility of integration that really matters.
Dawn Watson received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.
Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.
Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.
Reversed Phase HPLC for the Analysis of Biomolecules
November 15th 2016Biopharmaceuticals offer great hope in treating medical conditions which are currently poorly served, at best, by traditional pharmaceuticals. It is estimated that there are over 400 biopharmaceuticals in clinical trials for in excess of 200 disease areas. The enhanced complexity and variability that comes from the size of biopharmaceuticals, allied with the intricacy of the production process, mean chromatography is employed to a much greater extent during production and release testing. The following article will introduce the fundamentals of biopharmaceutical analysis and cover the use of reversed phase HPLC in the analysis of biomolecules. A subsequent article will detail the application of HILIC, IEX, and SEC chromatography for the analysis if biomolecules.